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Title:DOLOČANJE ACETILHOLINESTERAZE V HUMANIH KRVNIH VZORCIH
Authors:Birsa, Tjaša (Author)
Korte, Dorota (Mentor) More about this mentor... New window
Bergant Marušič, Martina (Mentor) More about this mentor... New window
Files:.pdf UNI_Birsa_Tjasa_i2018.pdf (1,65 MB)
 
Language:Slovenian
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:FZO - Faculty of Environmental Sciences
Abstract:V strokovni literaturi zasledimo encim acetilholinesterazo (AChE) v različnih raziskavah s področja okoljevarstva, medicine in veterine. Zmanjšana encimska aktivnost je lahko indikator onesnaženosti okolja z organofosfatnimi in karbamatnimi pesticidi, policikličnimi aromatskimi ogljikovodiki ter težkimi kovinami. Spremembe v encimski aktivnosti AChE pa so tudi biološki označevalec nekaterih nevrodegenerativnih bolezni pri človeku. V človeškem telesu se encim AChE primarno nahaja v živčnem sistemu, najdemo pa ga tudi v krvi, zlasti v membranah eritrocitov. V krvi je v večji meri prisoten tudi encim butirilholinesteraza (BChE), ki ravno tako spada v družino holinesteraz. Naš cilj je bil razviti metodo, ki bi omogočala specifično merjenje encimske aktivnosti AChE v okviru celotnih holinesteraz v krvnem serumu. Za merjenje encimske aktivnosti smo uporabili Ellmanovo metodo. Količino obarvanih produktov reakcije smo določali s spektroskopijo s toplotnimi lečami TLS in z UV-VIS spektrofotometrijo. Z različnimi koncentracijami čistega encima AChE smo najprej določili mejo zaznavnosti obeh metod (LOD). Detekcija s TLS je bila bolj občutljiva, saj je bil LOD 3,13-krat manjši kot pri detekciji z UV-VIS. Potem smo izmerili encimsko aktivnost celotnih holinesteraz AChE in BChE v serumskih vzorcih zdravih ljudi. Izračunana encimska aktivnost holinesteraz se je v analiziranih vzorcih gibala med 3004 mU mL-1 in 5698 mU mL-1. Po dodatku specifičnega inhibitorja BChE se je celotna encimska aktivnost znižala za najmanj 60 %, kar kaže, da večina holinesteraz v krvnem serumu pripada BChE. Preizkusili smo tudi metodo enojne imunske precipitacije, vendar se je le-ta izkazala za neuspešno pri ločevanju posameznih holinesteraz iz serumskih vzorcev. Na podlagi naših rezultatov smo prišli do zaključka, da je za merjenje encimske aktivnosti posameznih holinesteraz AChE in BChE v krvnem serumu najprimernejša uporaba specifičnih inhibitorjev, vendar bi bilo to metodo potrebno še dodatno optimizirati.
Keywords:acetilholinesteraza, krvni serum, nevrodegenerativne bolezni, spektroskopija s toplotnimi lečami, UV-VIS spektrofotometrija, imunska precipitacija.
Year of publishing:2018
Source:Nova Gorica
COBISS_ID:5264891 Link is opened in a new window
URN:URN:SI:UNG:REP:RTJ7P7UN
Views:1552
Downloads:88
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Secondary language

Language:English
Title:DETERMINATION OF ACETYLCHOLINESTERASE IN HUMAN BLOOD SAMPLES
Abstract:In the scientific literature, enzyme acetylcholinesterase (AChE) is discussed in array of topics, such as environmental, medical and veterinary sciences. Decrease in the enzymatic activity could be an indicator of an environmental pollution with organophosphate and carbamate pesticides, polycyclic organophospahic hydrocarbons or heavy metals. Changes in the enzymatic activity are also a biomarker of human neurodegenerative diseases. In human body, enzyme AChE can be found in the nerve system and blood, especially in the membrane of erythrocytes. High amounts of butyrylcholinesterase (BChE), another cholinesterase enzyme, can also be found in the human blood. Our aim was to develop a method that would enable specific determination of AChE enzymatic activity in the total cholinesterase activity of the blood. Ellman's method was used for the measurements of AChE activity. The quantity of the coloured products was measured with thermal lens spectroscopy (TLS) and UV-VIS spectroscopy. Firstly we have detemined the lower limit of detection (LOD) of the both methods. In the case of TLS method, LOD was 3,13 times lower, suggesting that this method is more sensible in comparison to UV-VIS spectroscopy. Afterwards we have measured the enzymatic activity of whole cholinesterases, AChE and BChE, in the human blood serum of healthy individuals. The cholinesterase activity was between 3004 mU mL-1 and 5698 mU mL-1. After we applied a specific enzymatic inhibitor of BChE, the whole enzymatic activity decreased for over 60 %, which shows that the majority of cholinesterase activity in the human blood serum was due to BChE. Immunodepletion is another technique that we used for the segregation of AChE and BChE activity in the blood serum. We have proved that one-time immunodeplation was not successful in the separation of a particular cholinesterase from the serum. Based on our results, we can conclude that the most suitable method for the specific measurements of the AChE and BChE activity in the human blood serum is the use of specific inhibitors. However, this method should be further optimized.
Keywords:acetylcholinesterase, blood serum, neurodegenerative disease, thermal lens spectrometry, UV-VIS spectrophotometry, immunoprecipitation


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